CELLULAR METABOLISM ASSAYS

Cell Viability, Cytotoxicity AND Proliferation Assays

Cell viability and cytotoxicity assays are used for drug and cosmetic ingredient screening, and cytotoxicity tests of chemicals. In vitro cytotoxicity testing provides a crucial means of ranking compounds for consideration in drug discovery. The choice of using a particular viability or cytotoxicity assay technology may be influenced by specific research goals.

In vitro methods have become a cornerstone of drug discovery and are widely applied both for screening and mechanistic studies. Whether researching cytotoxic, deleterious (toxic), or protective effects, the determination of concentrations that are cytotoxic to the model should be the primary step of in vitro testing.

  • Annexins are a family of calcium-dependent phospholipid-binding proteins which bind to phosphatidylserine (PS). Externalization of phosphatidylserine residues on the outer plasma membrane of apoptotic cells allows detection using Annexin V. Once the apoptotic cells are bound with labeled Annexin V, they can be visualized with fluorescent microscopy or cytometry.

 

  • The LDH Cytotoxicity Assay kit uses the formazan dye INT as a colorimetric indicator of lactate dehydrogenase enzyme activity.The Resazurin Cell Viability Assay is a fluorescent assay that detects cellular metabolic activity.

 

  • The Senescence Detection Kit measures activity of SA-β-gal in cells cultures by hydrolysis of X-gal, which results in the accumulation of a distinctive blue color in senescent cells.

 

  • The Sulforhodamine B (SRB) Cytotoxicity Assay is based on the ability of SRB to bind to protein components of cells that have been fixed to tissue culture plates to measure the number of cells and percent of cytotoxicity.

 

  • The PI/Cell Cycle Analysis Kit uses propidium iodide to fluorescently bind to DNA, which then determines the DNA content of the cells. Cells at different stages of the cell cycle can be distinguished by their DNA content.

 

  • Trypan Blue Solution, 0.4%, is used to stain cells to asses cell viability through the dye exclusion test.

 
Oxidative Stress and Enzyme Assays

Oxidative stress involves the chemistry of reactions of so- called reactive species derived from oxygen and nitrogen, particularly •OH, ONOO− and HOCl. The second mechanism of oxidative stress is aberrant redox signalling. Oxidants, particularly H2O2 generated by cells upon physiological stimulation, can act as second messengers.

To defend against oxidative injury, organisms have evolved defences primarily dependent upon antioxidant enzymes.

In normal physiological situations, Radical Oxygen Species (ROS) are constantly produced in our organism, where they even play several physiological roles, and their production is regulated by an efficient antioxidant defense (vitamins, oligoelements, proteins and enzymes) to prevent excessive cell damage.

All situations either physiological (prolonged exposure to pollution or sunlight, heavy consumption of alcohol and/or drugs, unbalanced physical activities, smoking, oxidant deficient diets…) or pathological  that will induce a deterioration in our antioxidant defense system will drive an overproduction of ROS in cells. The unstable nature of the latter makes them particularly reactive and capable of inflicting major cell damage by causing breaks and mutation in DNA, by inactivating proteins and enzymes, by oxidizing sugars, and by inducing lipid peroxidation among the polyunsaturated fatty acids of lipoproteins or of the plasma cell membrane.

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in Roles of Vascular Oxidative Stress and Nitric Oxide in the Pathogenesis of Atherosclerosis, Ulrich Förstermann, Circulation Research, Volume: 120, Issue: 4, Pages: 713-735, DOI: (10.1161/CIRCRESAHA.116.309326)

  • In the Catalase Activity Assay, catalase first reacts with H2O2 to producing water and oxygen. The unconverted H2O2 reacts 1:1 with the fluorogenic substrate to produce a highly fluorescent product.

  • The Reactive Oxygen Species (ROS) Detection Assay kit uses the cell permeant reagent Dichlorodihydrofluorescein-diacetate (H2DCFDA), a fluorogenic dye that measures hydroxyl, peroxyl and other (ROS) activity within the cell.

  • In the Superoxide (SOD) Activity Assay, SOD ions are generated from the conversion of xanthine and O2 to uric acid and H2O2 by Xanthine Oxidase (XO). The superoxide anion then coverts the tetrazolium salt WST-1 to the colored product WST-1 formazan. SOD activity in the experimental sample is measured as the percent inhibition of the rate of WST-1 formazan formation.

  • Oxidative stress causes decomposition of the unstable peroxides derived from polyunsaturated fatty acids resulting in the formation of malondialdehyde (MDA). The Thiobarbituric Acid Reactive Substances (TBARS) assay is based on the reaction of malondialdehyde (MDA) with thiobarbituric acid (TBA), which can be quantified colorimetrically.

Reporter Gene Assays
 
  • The FDG β-Galactosidase Assay quickly measures the levels of active β-galactosidase expressed in cells transfected with plasmids expressing Lac Z.

 

  • The Firefly & Renilla Dual Luciferase Assay kit exploits the differing biochemical requirements for luminescence of the firefly (Photinus pyralis) and sea pansy (Renilla reniformis) luciferase proteins. The kit allows the sequential quantitative measurement of both luciferase activities in a single protein extract.

 

  • The Firefly Luciferase Assay kit is designed for simple and efficient quantitation of firefly luciferase reporter enzyme activity from cultured cells with high sensitivity and linearity.

 

  • The ONPG B-Galactosidase Assay kit is a useful tool to quickly measure the levels of active β-galactosidase expressed in cells transfected with plasmids expressing Lac Z.

 

  • The Secreted Alkaline Phosphatase (SEAP) Reporter Gene Assay kit utilizes enzyme activity of alkaline phosphatase to dephosphorylate the chemiluminescent alkaline phosphatase substrate CSPD into an unstable dioxetane anion which decomposes and emits light.

 
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